The central theme and long-term objective of this project is to determine how various motifs in the CD3 cytoplasmic domains cooperate to control the cell biology (internalization, recycling & down-modulation) and signaling functions (T cell development & function) of the TCR. It is likely that certain motifs perform multiple functions (e.g. internalization & signaling), and that several motifs perform the same function (e.g. different ITAMs mediate signaling). Thus, a full understand of this complex and essential multichain receptor will not emerge until an integrated effort is undertaken to elucidate how CD3 performs and regulates these functions. Specific Aim 1: What residues mediate TCR:CD3 internalization and down-modulation? TCR:CD3 complexes will be transiently expressed on 293T cells using a panel of CD3 cytoplasmic domain mutants in the presence or absence of dominant active p56lck Flow cytometric and biochemical assays will be used to determine the effect of these mutations on TCR internalization and downmodulation. Yeast two-hybrid and immunoprecipitation experiments will be performed to determine which components of AP complex mediate these events, and to which residues in CD3 they interact. Specific Aim 2: Is TCR internalization an important physiological event for T cells? Using retroviral-mediated stem cell gene transfer, TCR expression will be restored in CD3gamma/delta KO mice using 2A-linked vectors containing wild-type and mutant CD3gamma/delta. The effect of preventing TCR internalization on T cell development, peripheral T cell phenotype, T cell function, cytokine production, and the response to influenza virus will be determined. Specific Aim 3: How do ITAM residues in the different CD3 chains cooperate to mediate T cell development and function? Retroviral-mediated stem cell gene transfer using a multi-cistronic 2A-linked vector will be used to restore TCR expression in mice lacking all four CD3 chains. Different combinations of TFAM mutants will be used to determine if the CD3 chains function in a qualitative and/or quantitative manner. The effect of these mutations on T cell development and function will be assessed as outlined in Aim 2.